A recent trend in cancer chemotherapy is the highly aggressive application of high-dose multiple drug treatment regimens at the earliest point of diagnosis. These protocols are limited by drug toxicity and severe physiological effects and patient fatalities are not uncommon. This situation has caused several members in the medical community to question whether the benefit/risk boundary has been exceeded with the agents currently available. Enhancement of the differential specificity of anticancer agents by selective targeting mechanisms might diminish such problems. The vitamin folic acid has attracted considerable attention as a potential means to selectively deliver covalently bound drug conjugates. Many human cancer cell lines have been found to have highly overexpressed membrane-associated folate receptor proteins which binds folic acid.
Previously, it has been shown that the natural receptor-mediated endocytosis pathway for the vitamin folic acid can be exploited to selectively and non-destructively deliver folate-conjugated small molecules, macromolecules, and drug carriers such as liposomes into cultured tumor cells. When folate is covalently linked to a folate molecule via its .gamma.-carboxyl moiety, the affinity of the covalent conjugate for the folate cell surface receptor remains essentially unaltered. Further, following binding to the cell surface receptor, the conjugated folate is internalized by the cell in much the same manner as the unmodified vitamin. Recycling of the folate receptor can then lead to further accumulation of the folate conjugates in such target cells.
Unfortunately, a major impediment in design of folate conjugates centers around synthesis of such compounds. Current practice simply involves treatment of the substrate of choice with folic acid or a folic acid analog and a dehydrating agent, such as DCC. This results in a mixture of both the inactive .alpha.-conjugate and the active .gamma.-conjugate often accompanied with the bis-functionalized derivative and/or recovered folic acid (see FIG. 1). Separation of these mixtures is often very difficult.
An alternative approach to the synthesis of differentially functionalized folic acid derivatives relies upon the acylation of monoesters of glutamic acid with pteroic acid. Unfortunately that strategy is compromised by the high cost of pteroic acid.
It is an object of the present invention to provide a method for regiospecific substitution of the .gamma.-carboxylic acid functional group of folic acid and related compounds.
There is also provided in the present invention novel pteroic acid derivatives. The glutamic acid moiety of folic acid derivatives is cyclized to form a pyroglutamate group. The pyroglutamate group is nucleophilically displaced to yield pteroic acid derivatives.
This invention also encompasses a method directed to preparing pteroic acid hydrazides by reacting corresponding pyroglutamates with hydrazine.
Another aspect the present invention encompasses a method of preparing .gamma.-substituted folic acid derivatives by reacting the corresponding pteroic azides with .gamma.-substituted glutamate esters.